Evaluation of in-vitro susceptibility of ß-lactam-resistant Gram-negative bacilli to ceftazidime-avibactam and ceftolozane-tazobactam from clinical samples of a general hospital in southern Brazil

ABSTRACT Background: The spread of carbapenemase- and extended-spectrum β-lactamase (ESBL)-producing gram-negative bacilli (GNB) represent a global public health threat that limits therapeutic options for hospitalized patients. This study aimed to evaluate the in-vitro susceptibility of β-lactam-resistant GNB to ceftazidime-avibactam (C/A) and ceftolozane-tazobactam (C/T), and investigate the molecular determinants of resistance. Methods: Overall, 101 clinical isolates of Enterobacterales and Pseudomonas aeruginosa collected from a general hospital in Brazil were analyzed. Susceptibility to the antimicrobial agents was evaluated using an automated method, and the minimum inhibitory concentrations (MIC50/90) of C/A and C/T were determined using Etest®. The β-lactamase-encoding genes were investigated using polymerase chain reaction. Results: High susceptibility to C/A and C/T was observed among ESBL-producing Enterobacterales (100% and 97.3% for CLSI and 83.8% for BRCAST, respectively) and carbapenem-resistant P. aeruginosa (92.3% and 87.2%, respectively). Carbapenemase-producing Klebsiella pneumoniae exhibited high resistance to C/T (80%- CLSI or 100%- BRCAST) but high susceptibility to C/A (93.4%). All carbapenem-resistant K. pneumoniae isolates were susceptible to C/A, whereas only one isolate was susceptible to C/T. Both antimicrobials were inactive against metallo-β-lactamase-producing K. pneumoniae isolates. Resistance genes were concomitantly identified in 44 (44.9%) isolates, with bla CTX-M and bla SHV being the most common. Conclusions: C/A and C/T were active against microorganisms with β-lactam-resistant phenotypes, except when resistance was mediated by metallo-β-lactamases. Most C/A- and C/T-resistant isolates concomitantly carried two or more β-lactamase-encoding genes (62.5% and 77.4%, respectively).


INTRODUCTION
Significant clinical and economic impacts are often reported because of bacterial resistance, since long hospital stays and the empirical use of different antimicrobial agents increase healthcare costs, as well as morbidity, and mortality rates 1 . The rapid spread of carbapenemase-and extended-spectrum β-lactamase (ESBL)-producing gram-negative bacilli (GNB) represents an important threat to global public health 2,3 and has limited the use of broadspectrum cephalosporins and carbapenems in hospitalized patients 1,4 .
In 2017, the World Health Organization (WHO) published a list of potentially critical multidrug-resistant microorganisms with global priority for the research and development of new antimicrobials, including carbapenem-resistant Pseudomonas aeruginosa and Enterobacterales resistant to carbapenems and thirdgeneration cephalosporins 5 . A few antimicrobial agents have been developed in recent years to combat infections caused by multidrug-resistant GNB 6,7 .
Ceftazidime-avibactam and ceftolozane-tazobactam were approved by the Food and Drug Administration (FDA) and the Brazilian Health Regulatory Agency (ANVISA) for the treatment of complicated intra-abdominal and urinary infections 8,9 . Ceftazidimeavibactam exerts in-vitro activity against clinical ESBL-producing isolates, including Ambler classes A (serine carbapenemases [KPC]), C (cephalosporinase-AmpC), and some class D enzymes (oxacillinases), but not metallo-β-lactamases (MβL) 9,10 . With the addition of avibactam, a β-lactamase inhibitor, ceftazidime tends to expand its activity against resistant strains 6 . Ceftolozane-tazobactam, which is currently approved for the treatment of hospital-acquired and mechanical ventilation-associated bacterial pneumonia 11 , is a combination of a fifth-generation cephalosporin and a known β-lactamase inhibitor. These agents together exert broad-spectrum activity against gramnegative bacteria, especially multidrug-resistant P. aeruginosa [12][13][14][15] .
Although recently approved for clinical use and despite its proven efficacy against GNB, resistance to ceftazidimeavibactam and ceftolozane-tazobactam has been reported in several countries 16 . In this context, the present study evaluating the in-vitro activity of these antimicrobial agents, as well as genotypic resistance markers, is important for optimizing their use and will also contribute to the understanding of the current epidemiological scenario.

Study characterization and selection of clinical isolates
This descriptive study focused on the phenotypic and molecular investigation of P. aeruginosa and Enterobacterales resistant to at least one carbapenem antibiotic or ESBL-producing antibiotic. The isolates were obtained sequentially from microbiological cultures of clinical samples collected from a general hospital in southern Brazil from January 2018. The clinical samples were subjected to routine procedures in the microbiology laboratory of the hospital for the identification of each microorganism, using the automated Microscan Walkaway Plus system (Beckman Coulter, USA) as well as Gram staining.

Phenotypic determination of antimicrobial susceptibility
The antimicrobial susceptibility profile was evaluated using the Kirby-Bauer disk diffusion method and the automated Microscan Walkaway Plus system (Beckman Coulter, USA) to determine the minimum inhibitory concentration (MIC) of each antimicrobial agent. Additionally, the MICs of ceftazidime-avibactam and ceftolozane-tazobactam were defined by a quantitative method using standardized Etest ® strips that contained an exponential concentration gradient. The concentration range used for both antimicrobials was 0.016/4-256/4 mg/L and the results were interpreted using the parameters of the Clinical and Laboratory Standards Institute (CLSI) and of the Brazilian Committee on Antimicrobial Susceptibility Testing (BRCAST).
The isolates were classified as ESBL producers based on the observation of a reduction in the inhibition halos for broadspectrum β-lactams in the antimicrobial susceptibility test and double-disk synergy test. Isolates were classified as resistant to carbapenems (CR) when resistance to meropenem, ertapenem, or imipenem was identified. The phenotypic detection of carbapenemases was performed using the enzymatic blocking method described in ANVISA Technical Note No. 01/2013 17 , which provides prevention and control measures for infections caused by multidrug-resistant Enterobacterales.

Extraction of bacterial DNA and investigation of target genes
Bacterial DNA was extracted from Müller-Hinton agar cultures using heat shock, as previously described 18 . To confirm the suitability of the extracted DNA for subsequent genotype analysis, the 16S rRNA gene was identified by the polymerase chain reaction (PCR) 19 .
The presence of the target genes was also investigated using PCR. All reactions were carried out in a final volume of 50 μL, using 50-500 ng of extracted DNA. The PCR-amplified products were subjected to electrophoresis on 1% agarose gel and compared with a standard.
The bla SHV and bla CTX-M genes were investigated in isolates with positive phenotypic tests for the presence of ESBL, using specific primers. The thermocycling conditions consisted of an initial denaturation step at 94 o C for 3 min, followed by 35 cycles of denaturation, annealing, and extension (1 min at 72 o C) for each gene, and a final extension at 72 °C for 10 min.
To investigate the carbapenemase-encoding bla OXA-48-like , bla NDM-1 , bla KPC , bla SPM-1 , bla VIM , and bla IMP genes, isolates showing phenotypic resistance to carbapenems were subjected to PCR consisting of an initial denaturation at 94 o C for 3 min, followed by specific thermocycling conditions specific for each target gene. Reference strains were used to confirm the effectiveness of the target gene detection methods.

Statistical analysis
The samples were obtained using convenience sampling. Data were analyzed using descriptive statistics, with calculation of absolute and relative frequencies. Categorical variables are expressed as absolute numbers and percentages.

In-vitro activity of ceftolozane-tazobactam
The in-vitro activity of ceftolozane-tazobactam against each group of microorganisms selected in this study is shown in Table 1. The ESBL-producing Enterobacterales and CR P. aeruginosa isolates showed high susceptibility. A high resistance rate was observed in the KPC-producing K. pneumoniae isolates. Two CR K. pneumoniae isolates were resistant. All CR isolates of the CESP group and MβL-producing K. pneumoniae were resistant to ceftolozane-tazobactam. Table 2 shows the in-vitro activity of ceftazidime-avibactam against each group of microorganisms selected in this study. All ESBL-producing Enterobacterales isolates were susceptible to ceftazidime-avibactam, showing the lowest MIC values compared with the other groups of microorganisms tested. A high susceptibility rate was observed for CR P. aeruginosa.

In-vitro activity of ceftazidime-avibactam
The resistance rate of KPC-producing K. pneumoniae was low. All CR K. pneumoniae isolates were susceptible, whereas the MβLproducing K. pneumoniae isolates were resistant. Two bacterial isolates from the CESP group (E. cloacae and S. marcescens) were susceptible, and one was resistant to ceftazidime-avibactam. Figure 1 summarizes the comparison of ceftazidime-avibactam and ceftolozane-tazobactam susceptibility profiles with the other antimicrobials tested in the different groups of microorganisms studied according to the CLSI breakpoints.

Genotypic resistance markers
The 16S rRNA gene was amplified in most of the isolates studied (97%, 98/101). The percentage of isolates positive for resistance genes was high (78.6%). Table 3 shows the distribution of the bacterial isolates according to the investigated genotypic resistance markers. All the tested isolates were negative for bla SPM-1 , bla OXA-48-like , and bla IMP .
Coexistence of resistance genes was observed in 44 (44.9%) isolates. The most prevalent combinations were bla KPC +bla CTX-M +bla SHV in KPC-producing K. pneumoniae, and bla CTX-M +bla SHV in ESBL-producing Escherichia coli. Most isolates with phenotypic resistance to ceftazidime-avibactam and ceftolozane-tazobactam concomitantly carried two or more β-lactamase-encoding genes ( Table 4). The bla CTX-M , bla SHV , and bla KPC were most frequently detected in isolates resistant to ceftolozane-tazobactam, whereas bla SHV , bla CTX-M , and bla NDM-1 were most frequently detected in isolates resistant to ceftazidime-avibactam.

DISCUSSION
The increasing incidence of bacterial strains isolated from clinical samples that produce carbapenemases, enzymes capable of inactivating carbapenems, and most β-lactams 20 represents the greatest challenge of antibiotic therapy in recent years 2,6 . Enterobacterales and P. aeruginosa are the main causative agents of severe infections associated with antibiotic resistance resulting from chromosomal mutations and the transfer of plasmid-mediated resistance 21 . Such clinical isolates commonly produce carbapenemases and exhibit multidrug resistance and pan-resistance phenotypes 22 .  Studies have been conducted in different countries to evaluate in-vitro susceptibility to ceftazidime-avibactam and ceftolozanetazobactam, and bacterial resistance to these antimicrobial agents has been reported in hospitalized patients with or without previous treatment 16 . Furthermore, the combination of resistance mechanisms can significantly increase the MIC of ceftazidimeavibactam and ceftolozane-tazobactam 16,23 .
The ability of ceftazidime-avibactam to inhibit KPC-type β-lactamases has attracted global interest. In China, Cui et al. evaluated 347 KPC-producing K. pneumoniae isolates collected from patients without previous treatment with only 12 (3.5%) isolates showing reduced susceptibility to ceftazidime-avibactam 24 . In Brazil, Rossi et al. found that among 30 selected K. pneumoniae isolates that were not susceptible to meropenem and positive for bla KPC , only one (3.3%) was resistant to ceftazidime combined with avibactam 9 . Similarly, in our study, the presence of bla KPC did not influence ceftazidime-avibactam susceptibility, and the resistance rate observed (6.6%; 1/15) was consistent with the global surveillance results of carbapenem-resistant and bla KPC -carrying K. pneumoniae 10 .
Jonge et al. characterized the in vitro activity of ceftazidimeavibactam against 961 meropenem-non-susceptible Enterobacterales isolates from Europe, Asia, Latin America, and the Middle East using a global antimicrobial resistance surveillance program. The authors evaluated 145 MβL-producing isolates and detected a ceftazidime-avibactam resistance rate of 96.6% 10 . Similarly, all MβL-producing K. pneumoniae isolates in this study were resistant to ceftazidime-avibactam. Thus, ceftazidime-avibactam is a potent agent against carbapenem-resistant Enterobacterales, except for isolates in which resistance is mediated by MβL.
According to international reports, the resistance rates of P. aeruginosa to ceftazidime-avibactam are higher than those reported for Enterobacterales, ranging from 2.9 to 18%, whereas these rates can reach 50% in isolates resistant to carbapenems 16 . Although this study investigated carbapenem-resistant P. aeruginosa isolates, the rate of ceftazidime-avibactam resistance was low (7.7%; 3/39). This might be related to the absence of carbapenemase-encoding genes in most of the isolates investigated, suggesting that the detected phenotypic resistance to carbapenems is associated with other pseudomonal resistance mechanisms not analyzed here/in this study.
Studies have reported that bacteremia caused by ESBLproducing Enterobacterales is associated with higher rates of treatment failure and patient mortality when compared to bacteremia caused by non ESBL-producing strains 1,25 . Various authors have emphasized that ESBL-producing Enterobacterales strains are not associated with resistance to ceftazidimeavibactam 6,7,10,15,25,26 , which was also demonstrated in our study.
López-Calleja et al. analyzed the multidrug-resistant and extensively drug-resistant non-MβL-producing P. aeruginosa isolates collected in Spain and reported 92.2% susceptibility to ceftolozane-tazobactam, which was the second most active antimicrobial agent after colistin 27 ; this was also observed in our study (87.2%). In particular, we did not identify bla NDM-1 or bla IMP genes in carbapenem-resistant P. aeruginosa isolates, and the bla VIM gene detected in only one isolate was not associated with the ceftolozane-tazobactam resistance phenotype. In contrast, Teo et al. highlighted the importance of geographic variation in antimicrobial activity. The authors reported much lower   Presence of β-lactamase-encoding genes and phenotypic susceptibility to ceftazidime-avibactam (C/A) and ceftolozane-tazobactam (C/T) in the isolates studied. susceptibility rates of P. aeruginosa to ceftolozane-tazobactam (37.9%), which was associated with the presence of MβL, compatible with local molecular epidemiology 28 .

Phenotypic resistance Species Isolates (n) β-Lactamase genes
Tuon et al. evaluated 673 GNB isolates collected from different Brazilian centers and found rates of in-vitro susceptibility to ceftolozane-tazobactam ranging from 40.4% to 94.9%. The susceptibility rate of K. pneumoniae to ceftolozane-tazobactam was low (40.4%) because of the high incidence of KPC-type carbapenemases in Brazil, an enzyme that catalyzes the hydrolysis of ceftolozane 29 . In our study, the susceptibility rate of KPCproducing K. pneumoniae to ceftolozane-tazobactam was even lower (13.3%). This finding might be explained by the identification of bla KPC gene in all isolates tested and the concomitant presence of ESBL-encoding genes. Additionally, two isolates carrying more than one carbapenemase-and ESBL-encoding gene (bla KPC +bla CTX-M +bla SHV +bla NDM-1 and bla KPC +bla CTX-M +bla SHV +bla VIM ) were identified in association with ceftolozane-tazobactam resistance phenotypes. Regarding MβL-producing K. pneumoniae, all isolates were resistant to ceftolozane-tazobactam and most of them carried bla NDM-1 , suggesting that this agent should be used with caution in empirical therapies.
In contrast, we found high rates of in-vitro ceftolozane-tazobactam susceptibility among ESBL-producing Enterobacterales. This finding in consistent with a study that evaluated 21,952 Enterobacterales isolates from 51 countries and found that ceftolozanetazobactam inhibited 82.4% of ESBL-producing isolates 15 .
This study had some limitations. The number of isolates evaluated was relatively small, and the study was conducted at a single hospital. However, the study used clinical isolates selected in recent years to better reflect the current epidemiological scenario. Therefore, we recommend multicenter studies using a phenotypic and genotypic approach and a greater number of multidrugresistant isolates for better understanding of the local molecular epidemiology and for the detection of resistance to ceftazidimeavibactam and ceftolozane-tazobactam in different regions of Brazil.
In conclusion, we found high susceptibility rates to ceftazidimeavibactam and ceftolozane-tazobactam among ESBL-producing Enterobacterales and carbapenem-resistant Pseudomonas aeruginosa. In contrast, carbapenemase-producing Klebsiella pneumoniae exhibited high resistance to ceftolozane-tazobactam but high susceptibility to ceftazidime-avibactam. Our results obtained in-vitro confirmed that ceftazidime-avibactam and ceftolozane-tazobactam were active against microorganisms with β-lactam resistance phenotypes, except when resistance was mediated by metallo-β-lactamases. Additionally, most ceftazidimeavibactam-and ceftolozane-tazobactam-resistant isolates concomitantly carried two or more β-lactamase-encoding genes.